Active transport proteins are involved in a multitude of cellular reactions, facilitating the passage of specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. These integral proteins establish the basis for membrane function and thus make possible the generation of energy and transport of essential nutrients in all forms of life. Furthermore, aberrant function of membrane proteins is causally implicated in many human diseases. Understanding the dynamics of membrane protein structure and function therefore constitutes a critical objective for basic and medical research. During the initial grant cycle, we solved the structure of the Na+/galactose symporter from Vibrio parahaemolyticus (vSGLT) in the inward-occluded conformation. More recently, we solved an inward-open conformation of vSGLT. Together, these structures (in conjunction with biochemical and molecular dynamics simulations) show Na+ exit causes a reorientation of transmembrane helix 1 that opens an inner molecular "gate" permiting galactose release. This renewal application will capitalize on the gains made during the first cycle by combining crystallography, state-of-the-art spectroscopy and diffuse X-ray diffraction techniques to measure intricate movements of entire regions of the protein. These approaches, coupled with physiological assays and molecular dynamics simulations, will provide insights into membrane transport in real-time. This proposal has four primary goals: 1) we will use inhibitors and mutants of essential residues to alter the conformational equilibrium of vSGLT and resolve new structures;2) we will monitor substrate-induced conformational changes using double electron-electron resonance (DEER);3) we will capture real-time inter- atomic conformational changes using Time Resolved Small- and Wide-Angle X-ray Scattering (TR-S/WAXS);and 4) we will determine structures of the pharmaceutically relevant human members of the SSS family. Each aim on it own is capable of producing exciting results, but when these complementary approaches are merged together they will provide a more complete picture of Na+ and sugar co-transport. PUBLIC HEALTH RELEVANCE: Active transport proteins are involved in a multitude of cellular responses and important components in human health and disease. The functional and dynamical properties of these proteins are fundamentally coupled to their three-dimensional structures which are modulated at the atomic level over a broad range of time scales. These studies will provide a complete atomic resolution mechanism of the sodium galactose transporter as it proceeds through its reaction cycle.